Methods and compositions for treatment of penile defects

ABSTRACT

A composition for use in treating penile defects, including erectile dysfunction and Peyronie&#39;s disease, is provided. The composition includes adipose tissue and stem cells. The stem cells may be derived from adipose tissue. In embodiments the composition includes additional additives such as growth factors, anti-inflammatories, antioxidants, compositions useful for wound healing, and collagenases. Also provided is a method of treating a penile defect in a patient. The method includes providing a composition containing adipose tissue and stem cells, and implanting that composition into a patient in need of treatment for a penile defect. In some embodiments the patient has Peyronie&#39;s disease. In other embodiments, the patient has erectile dysfunction.

RELATED APPLICATIONS

This application is a division of co-pending U.S. application Ser. No.15/158,101 filed on May 18, 2016 which is a division of co-pending U.S.application Ser. No. 14/363,142 filed on Jun. 5, 2014 which is theUnited States national phase of International Application No.PCT/US2013/023830 filed Jan. 30, 2013 and claims priority to U.S.Provisional Application No. 61/592,108 filed Jan. 30, 2012, thedisclosures of which are hereby incorporated in their entirety byreference.

TECHNICAL FIELD

The present invention relates to compositions for use in medicalprocedures and methods for use thereof. More specifically, the inventionrelates to compositions of tissue, including stem cells, useful forregenerating tissue in disease, specifically in penile disease anddefects.

BACKGROUND OF THE INVENTION

The Use of stem cells has grown exponentially over the past two decadesas methods of isolating and purifying these cells has become simpler andmore cost-effective. In particular, with the advent of the ability toobtain adult stem cells, as opposed to politically-charged embryonicstem cells, work in the field has blossomed. Stem cells have beensuggested as a cure for ailments ranging from Parkinson's disease(replacing dying dopaminergic cells of the substantia nigra) tomyocardial infarctions and cardiomyopathy. Gimble J M et al.Adipose-derived stem cells for regenerative medicine. Circulation Res.2007; 100: 1249-1260; Kondo K et al. Implantation of adipose-derivedregenerative cells enhances ischemia-induced angiogenesis.Arteriosclerosis, Thrombosis, and Vascular Biol. 2009; 29: 61-66.

Other defects and diseases may also be candidates for stem cellintervention. One area of interest is that of penile defects, such aserectile dysfunction (ED) and Peyronie's disease. At least some forms ofED trace their etiology to vascular deficits in the penile tissue.Pharmaceutical compositions for treating ED do exist; however, a needexists for a treatment that does not require use of medicaments. Atleast one group has reported success in the use of stem cells derivedfrom bone marrow to reverse ED in an animal model. Kendirci M et al.Transplantation of non-hematopoietic adult bone marrow stem/progenitorcells isolated by the p75 nerve growth factor receptor into the penisrescues ED in a rat model of cavernous nerve injury. J. Urol. 2010;184(4): 1560-1566. Another group has seen success in usingmuscle-derived stem cells in a rat model of ED. Woo J C et al.Transplantation of muscle-derived stem cells into the corpus cavernosumrestores erectile function in a rat model of cavernous nerve injury.Korean J. Urol. 2011; 52: 359-363.

Peyronie's disease is an ailment involving the growth of fibrous plaquesin the soft tissue of the penis. These plaques grow in the tunicaalbuginea, a region of connective tissue that is part of the moregeneral connective tissue of Bucks fascia. The tunica albuginea is anarea surrounding the corpora cavernosa. The disease results in pain, ED,and alteration of penis shape, and may be caused by disorganizedcollagen fibers. Treatments for the disease currently includeadministration of corticosteroids to the plaques, radiation therapy, andvitamin E Pharmaceuticals for treatment exist; however, the use ofmedicaments is considered controversial in the art, and these compoundshave not met with sustained success. Hauck E W et al. A criticalanalysis of nonsurgical treatment of Peyronie's disease. EuropeanUrology 2006; 49(6): 987-97.

In light of the above, a need exists in the art for a composition ofstem cells that is easy to obtain and isolate and a method for use ofstem cells in treating penile defects

SUMMARY OF THE INVENTION

The present invention provides a composition, the composition comprisingadipose tissue and at least one isolated stem cell. In non-limitingembodiments, the at least one stem cell is placental or embryonic inorigin. In other non-limiting embodiments, the at least one stem cell isa mesenchymal stem cell. In certain non-limiting embodiments, the atleast one stem cell is an adipose-derived stem and regenerative cell(ADRC). In further non-limiting embodiments, the stem cell and adiposetissue are derived from a donor. In certain further non-limitingembodiments, the stem cell is an ADRC and it is derived from a donor. Infurther non-limiting embodiments the at least one stem cell isgenetically engineered.

In additional non-limiting embodiments, the composition includes atleast one additional additive. The additive may be a compound useful inwound healing, in decreasing inflammation, in breaking down collagen, orin promoting angiogenesis and/or vasculogenesis. In further non-limitingembodiments the composition includes a growth scaffold.

The present invention also provides a method of treating a penile defectin a subject, comprising providing a composition comprising at least oneisolated stem cell, and implanting the composition with the penis of thesubject. In certain non-limiting embodiments, the composition comprisesadipose tissue and stem cells, which may be derived from the subject. Innon-limiting embodiments, the at least one stem cell is placental orembryonic in origin. In other non-limiting embodiments, the at least onestem cell is a mesenchymal stem cell. In certain further non-limitingembodiments, the at least one stem cell is an ADRC. In furthernon-limiting embodiments, the adipose tissue and ADRC are derived fromthe subject. In non-limiting embodiments, the composition is implantedinto the subject's penis. In further non-limiting embodiments, thecomposition is implanted into or around the subject's corpus cavernosum.In another non-limiting embodiment, the composition is implanted into oraround the subject's tunica albuginea. In certain embodiments thecomposition is injected into the tissue of interest. In othernon-limiting embodiments the composition is injected or infused into anyartery that feeds penile tissue. In certain non-limiting embodiments,the composition is injected or infused into the corporal arteries orinternal pudendal arteries.

In additional embodiments, the composition that is implanted includes atleast one additional additive. The additive may be a compound useful inwound healing, in decreasing inflammation, in breaking down collagen, orin promoting angiogenesis and/or vasculogenesis. In further non-limitingembodiments the composition includes an implantable or injectable growthscaffold.

The present invention also provides a method of treating Peyronie'sdisease by injecting a composition comprising at least one mesenchymalstem cell, at least one component of extracellular matrix, and at leastone growth factor, and injecting that composition into the tunicaalbuginea of a subject in need of treatment for Peyronie's disease.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following description is merely exemplary in nature and is in no wayintended to limit the invention, its application, or uses.

Provided herein is a composition, the composition compromising adiposetissue and at least one isolated stem cell.

In certain non-limiting embodiments, the at least one stem cell is amesenchymal stem cell, preferably an adipose-derived stem andregenerative cell (ADRC). In further non-limiting embodiments, the stemcell and adipose tissue are derived from a donor. In certain furthernon-limiting embodiments, the stem cell is an ADRC and it is derivedfrom a donor. In further non-limiting embodiments the at least one stemcell is genetically engineered.

In certain non-limiting embodiments, the composition includes at leastone additional additive. The additive may be a compound useful in woundhealing, in decreasing inflammation, in breaking down collagen, or inpromoting angiogenesis and/or vasculogenesis. In further non-limitingembodiments the composition includes a growth scaffold.

The present invention also provides a method of treating a penile defectin a subject, comprising providing a composition comprising at least oneisolated stem cell, and implanting the composition with the subject.

In certain non-limiting embodiments, the composition includes adiposetissue and stem cells, which may be derived from the subject. In certainfurther non-limiting embodiments, the at least one stem cell is amesenchymal stem cell, preferably an adipose-derived stem andregenerative cell (ADRC). In further non-limiting embodiments, theadipose tissue and ADRC are derived from the subject. In non-limitingembodiments, the composition is implanted into the subject's penis. Infurther non-limiting embodiments, the composition is implanted into oraround at least one of the subject's corpus cavernosum. In anothernon-limiting embodiment, the composition is implanted into or around thesubject's tunica albuginea. In other non-limiting embodiments thecomposition is injected or infused into any artery that feeds peniletissue. In certain non-limiting embodiments, the composition is injectedor infused into the corporal arteries or internal pudendal arteries.

In additional embodiments, the composition that is implanted includes atleast one additional additive. The additive may be a compound useful inwound healing, in decreasing inflammation, in breaking down collagen, orin promoting angiogenesis and/or vasculogenesis. In further non-limitingembodiments the composition includes an implantable or injectable growthscaffold.

While particular focus is on the use of ADRCs in the composition andmethod described herein, any type of stem cell, defined below, may beused to advantageously promote vasculogenesis and wound healing inpenile defects. These properties function to address defects anddiseases such as ED. Additionally, ADRCs may be provided directly toplaques in Peyronie's disease, stimulating wound healing. ADRCs arepreferable for stem cell derived treatments of these defects because oftheir ability to stimulate vasculogenesis and to produce organizedcollagen. Thomas J. Stem cells repair damaged corneas in mice. HealingWell. Apr. 10, 2009, available athttp://news.healingwell.com/index.php?p=news1&id=625948.

Definitions

For convenience, certain terms employed in the entire application(including the specification, examples, and appended claims) arecollected here. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs.

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines or type of stem cell,constructs, additives, and reagents described herein. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention which will be limited only by theappended claims.

The term “stem cell” refers to any multipotent or pluripotent cell,traditional stem cells, progenitor cells, preprogenitor cells, andreserve cells. The term is used interchangeably with and may meanprogenitor cell. The stem cell may be derived from an adult organism orfrom a cell line, or from an embryonic organism. Exemplary stem cellsinclude embryonic stem cells, adult stem cells, pluripotent stem cells,neural stem cells, liver stem cells, muscle stem cells, muscle precursorstem cells, endothelial progenitor cells, bone marrow stem cells,chondrogenic stem cells, lymphoid stem cells, mesenchymal stem cells,hematopoietic stem cells, and the like. Descriptions of stem cells,including method for isolating and culturing them, may be found in,among other places, Embryonic Stem Cells, Methods and Protocols, Turksened., Humana Press, 2002.

The term “adult” as used herein refers to any non-embryonic organism.For example the term “adult adipose-derived regenerative cell,” refersto an adipose-derived regenerative cell, other than that obtained froman embryo.

The term “embryo” as used herein refers to any multicellular diploideukaryote during development, until birth or hatching. The term“embryonic stem cell” refers to a pluripotent cell derived from theinner cell mass of a blastocyst.

The term “mesenchymal stem cell” refers to any multipotent stromal cellderived from, for example and without limitation, umbilical cord blood,adipose tissue, muscle, corneal stroma, and dental pulp that candifferentiate into cells such as, including but not limited to,osteoblasts, chondrocytes, and adipocytes.

The term “adipose-derived regenerative cell” (ADRC) is usedinterchangeably with adipose stem cells (ASC) herein and refers to adultcells that originate from adipose tissue. ADRC are a heterologouspopulation of cells comprising at least one of the following populationof cells; adult stem cells, vascular endothelial cells, vascular smoothmuscle cells, endothelial cells, mesenchymal stem cells, fibroblasts,pericytes and additional other cell types.

In some embodiments, ADRC refers to a substantially pure population ofadipose-derived stem cells. ADRC can be easily harvested from adiposetissue and are substantially free of adipocytes and red blood cells andclonal populations of connective tissue stem cells. The stromal vascularfraction cells are substantially devoid of extracellular matrix materialfrom adipose tissue. ADRC may also be referred to as adipose-derivedstem/stromal cells (ASCs), adipose-derived adult stem (ADAS) cells,adipose-derived adult stromal cells, adipose-derived stromal cells,adipose stromal cells, adipose mesenchymal cells, adipose-derivedmesenchymal stem cells, lipoblasts, pericytes, preadipocytes, andprocessed lipoaspirate cells.

The term “adipose” as used herein refers to any fat tissue from asubject. The terms “adipose” and “adipose tissue” are usedinterchangeably herein. The adipose tissue may be brown fat, white fator yellow fat or white adipose tissue, derived from subcutaneous,omental/visceral, mammary, gonadal, or other adipose tissue site. Theadipose tissue has adipocytes and stroma. Adipose tissue is foundthroughout the body of an animal. For example, in mammals, adiposetissue is present in the omentum, bone marrow, subcutaneous space, andsurrounding most organs. Such cells may comprise a primary cell cultureor an immortalized cell line. The adipose tissue may be from anyorganism having fat tissue.

Preferably, the adipose tissue is human; most preferably, the adiposetissue is derived from the individual in need of treatment for a peniledefect. A convenient source of adipose tissue is from liposuctionsurgery, however, the source of adipose tissue or the method ofisolation of adipose tissue is not critical to the invention, andacquisition of adipose tissue by any means may adequately provide tissueand stem cells for the present invention.

The term “tissue” as used herein is a broad term that is applied to anygroup of cells that perform specific functions, and includes in someinstances whole organs and/or part of organs. A tissue need not form alayer, and thus encompasses a wide range of tissue, including adiposetissue derived from any source in an organism. Preferably, the tissue isderived from a mammal Most preferably, the tissue is derived from theindividual in need of treatment for a penile defect.

The term “implant” as used herein refers to any method for transferringa population of cells or cell mass into a subject, including by surgicalimplantation (incision into the tissue of interest and depositiontherein) and injection by a syringe, needle, cannula, or the like of anysuitable gauge. An implant as used herein can comprise geneticallymodified cells, as well as cells differentiated from other cells, suchas stem cells, progenitors, and the like, as well as adipose cells ortissue.

The term “corpus cavernosum” of the penis refers to one of a pair ofsponge-like regions of erectile tissue which contain most of the bloodin the penis during penile erection. Generally, the two corpuscavernosum and a corpus spongiosum are three expandable erectile tissuesalong the length of the penis which fill with blood during erection. Theterm “corpus” is used interchangeably herein with corporal, corporealand corporic, which are terms used to describe tissues which are derivedfrom the corpora cavernosum or which can be developed, differentiated,or altered by natural or artificial means into corpora cavernosumtissue. The term “cavernosum” is used interchangeably herein ascavernae, corporum, cavernosum, or cavernosorum penis, and refers to thecaverns of corpora cavernosa (or one of the two corpus cavernosum) ofthe penis or the dilatable spaces within the corpus cavernosum of thepenis, which fill with blood and become distended with erection.

The term “tunica albuginea” refers to the fibrous tissue covering, orenveloping, the corpora cavernosa of the penis. This tissue consists ofelastin and collagen. The term “Buck's fascia” refers to the layer offascia covering the penis, including the tunica albuginea.

The terms “subject” and “individual” are used interchangeably herein,and refer to an animal, for example a human, from whom a adipose tissueand stem cells, for example ADRC can be harvested, or a subject intowhom tissue can be transplanted for treatment, for example treatment forpenile defects, using the compositions and methods described herein. Fortreatment of conditions or disease states which are specific for aspecific animal such as a human subject, the term “subject” refers tothat specific animal. In some embodiments, the subject is a humansubject. It is possible in embodiments of this invention that recipientsubjects are of a different mammalian subject than the donor subject.

DETAILED DESCRIPTION OF THE INVENTION

The Compositions and Methods for Treatment of Penile Defects, Peyronie'sDisease, Erectile Dysfunction

One aspect of the present invention provides for a composition. Thecomposition comprises adipose tissue and at least one isolated stemcell. The composition is one that is suitable for implantation into asubject, for example an animal, preferably a mammal. In a preferrednon-limiting embodiment, the composition is suitable for implantationinto a human. In a further preferred non-limiting embodiment, theadipose tissue and at least one stem cell of the composition aresuitable for implantation into a human because they are derived from thesame human subject.

The at least one stem cell may be any type of stem cell, including butnot limited to mesenchymal stem cells. The stem cells may be obtainedfrom any suitable source, for example, and without limitation, stemcells may be obtained from adult or embryonic sources, from bone marrow,from placental tissue, from umbilical cord blood, and from adiposetissue. In preferred, non-limiting embodiments, the stem cell isobtained from adipose tissue, and is an adipose-derived regenerativecell (ADRC).

In general, adipose tissue may be collected in any suitable manner. Forexample, in non-limiting embodiments, adipose tissue is obtained throughdry liposuction, wet liposuction, super-wet liposuction, tumescentliposuction, power-assisted liposuction, laser-assisted liposuction, orthe like.

Once harvested by any suitable method, adipose tissue may be processedby any known method. The following methods are to be consideredexemplary, and non-limiting. In a preferred, non-limiting embodiment,adipose tissue is processed using the StemSource® Cellbank or CytoriCelution® System. The Cytori procedure and system therefore aredisclosed in detail in U.S. Pat. Nos. 7,501,115 and 7,687,059. Briefly,adipose tissue is harvested from a subject. In a preferred, non-limitingembodiment of the current invention, the subject is an individual inneed of treatment for a penile defect. The tissue is processed to removemature adipocytes and connective tissue from the sample. Processing mayoccur within a system having a tissue collection port, filter disposedwithin the system, and a mixing container for holding the processedsample. The filter works to remove the unwanted cells and non-cellularmaterials from the sample, enriching the proportion of ADRCs in thefiltered sample.

The Cytori system has successfully been used in providing ADRCs forbreast tissue implantation for reconstruction after radical lumpectomy,proving the ability of stem cells isolated by this method to generatetissue of a single type (adipocytes, or fat). Similarly, in treatment ofpenile defects, only one type of tissue need be generated. As such, theCytori method and system is optimal for use in preparing the compositionof the current invention.

With regard to use of the Cytori system and method itself, processing isaccomplished by washing and disaggregating the tissue to reduce thepresence of free lipids and blood elements. Processing may or may notalso include a rinsing step; the step conducted using isotonic saline orany other suitable physiologic solution known to those skilled in theart.

Remaining tissue is then disaggregated using enzyme degradation ormechanical disaggregation. The cells and solution are then centrifugedto separate cells, including ADRC, from the degradation solution. Thecells form a pellet in the centrifuge, and the pellet can be frozen forstorage or resuspended in another solution, for example a buffer, foruse in medical treatments such as those recited herein.

The concentration of stem cells obtained by this method may vary, buttypically approximately 0.1% of the cells in the pellet are stem cells.Greater percentages may be obtained by varying the above method, forexample by use of adherence protocols such as described in Berdel W E etal., Purification of human monocytes by adherence to polymericfluorocarbon. Immunobiology 1982 163(5): 511-520, or by separation onthe basis of cell-surface markers, for example on the basis of markerspresent on differentiated cells, such as CD34 and the like, or byselecting based on markers expressed on progenitor cells, such as CD90and the like. In a preferred, non-limiting embodiment, the percentage ofstem cells present in the pellet is between approximately 2% and 12%.

For embodiments where the individual to be treated is not the donor ofthe adipose tissue and at least one stem cell, the composition mayfurther comprise immunosuppressive agents designed to prevent rejectionof the composition once implanted Immunosuppressive agents may beselected from glucocorticoids, cytostatics, antibodies, pharmaceuticalssuch as tacrolimus, ciclosporin, sirolimus, interferons, opioids,mycophenolic acid, fingolimod, and myriocin.

In other non-limiting embodiments adipose tissue is derived fromlipoaspirate (i.e. from liposuction), is washed, exposed to acollagenase digest at 37 degrees Celsius, and centrifuged.

In a non-limiting embodiment, ADRCs are isolated from adipose tissue bytriplicate washing in 01. mol/L phosphate-buffered saline (PBS), pH=7.4.Tissue is then minced and digested in 0.1% collagenase type I at 37degrees Celsius for 45 minutes. Afterwards, the solution is filtered andcentrifuged at 800 g for eight minutes. Supernatant is removed and cellsare resuspended in Modified Eagle's Medium (MEM) supplemented with 10%fetal bovine serum (FBS). Cells are then plated and incubated at 37degrees Celsius.

ADRCs are harvested from the growth by flow cytometry according totraditional flow cytometry methods, for example as disclosed inAlexander C M et al. Separating stem cells by flow cytometry: reducingvariability for solid tissues. Cell Stem Cell 2009; 5: 579-83 and OnlineSupplemental Material. Cells are washed in buffer (FBS) and incubatedfor 30 minutes in FBS with antibodies against CD105, CD90, CD34, and/orCD45. The ADRCs are then combined with adipose cells in a compositionfor injection.

In non-limiting embodiments, the ADRCs are processed by washing in PBSfollowed by ECM digestion in 0.075% collagenase for 30 minutes at 37degrees Celsius. Digestion is neutralized using Dulbecco's ModifiedEagle Medium (DMEM) containing 10% FBS. Digested tissue is thencentrifuged at 1200 g for 10 minutes to obtain a cell pellet. The pelletis then resuspended and filtered, and cells are plated. Cell media isremoved 12-18 hours later and fresh media provided. Cells are incubatedon ice with antibodies to human CD11b, CD18, CD31, CD34, CD38, CD44,CD45, CD54, CD62L, CD90, CD105, CD106, CD1 17, CD133, CD144, CD166,and/or CD271. Cells are then washed and flow cytometry, for examplefluorescence-activated cell sorting, is performed according totraditional, known methods, for example as disclosed in Alexander C M etal. Separating stem cells by flow cytometry: reducing variability forsolid tissues. Cell Stem Cell 2009; 5: 579-83 and Online SupplementalMaterial.

While specific ADRC isolation procedures are provided herein, theinvention described should not be limited to those procedures. It shouldbe apparent to those skilled in the art that other means of isolatingstem cells, in particular ADRC cells, are possible and may be usedinterchangeably with the current invention.

While the process for obtaining adipose tissue and ASCs is described indetail and is a preferred, non-limiting embodiment, other sources ofstem cells are within the scope and spirit of the present invention.

In non-limiting embodiments, the stem cell used in the presentcomposition is a mesenchymal stem cell isolated from bone marrow. Incertain preferred non-limiting embodiments, the bone marrow is that ofthe patient in need of treatment for a penile defect. Such stem cellsmay be isolated in accordance with the following protocol, which is tobe considered exemplary and non-limiting.

Bone marrow tissue may be obtained from the head of the femur, bytransversally segmenting the femoral head to expose trabecular, orcancellous, bone. This trabecular bone may be extracted with successive,serial washes of phosphate-buffered saline (PBS). The solution of PBSand trabecular bone may then be filtered through a filter of anysuitable pore diameter, for example and without limitation a 40, 70, or100 um cell strainer. The solution may then be centrifuged at 400 g for10 minutes to form a pellet of cells, the supernatant aspirated, and thepellet resuspended in media containing DMEM supplemented with 10% FBSand 1% streptomycin or penicillin, or both. Cells may then be cultured,and then washed with PBS to remove non-adherent cells. The obtainedcells may be further expanded by repeating the incubation and washingsteps until a suitable volume of cells is obtained. Medium may besupplemented with heparin and growth factors, if necessary.

In other non-limiting embodiments, the stem cells may be obtained fromplacental tissue. Stem cells from this source may be obtained by anymethod known to those of skill in the art. In certain non-limitingembodiments, stem cells may be obtained according to the followingprocedure. Tissue samples from placental tissue may be washed in PBSplus antibiotic solution (200 U/mL penicillin and/or 200 mg/mLstreptomycin). The amnion is separated from chorion through finedissection. Small pieces of both membranes are minced and subjected toenzymatic digestion to obtain a mesenchymal population of cells. Theresulting cells are seeded in 25 cm2 culture flasks with 5 mL of DMEMwith 20% FBS and antibiotics (penicillin 100 U/mL and/or streptomycin100 mg/mL), and incubated at 37 degrees Celsius. Nonadherent cells areremoved with changes of medium (DMEM plus 10% FBS). The obtained cellsmay be further expanded by repeating the incubation and washing stepsuntil a suitable volume of cells is obtained. Medium may be supplementedwith heparin and growth factors, if necessary.

In addition to the protocols described herein, stem cells may beobtained from any suitable commercial source for use in the compositionof the present invention. In non-limiting embodiments, mesenchymal stemcells from placental tissue provided under the trade name Ovation® byOsiris Therapeutics, Inc. may be used. This product is preferred becauseit includes components of extracellular matrix (ECM) and growth factors.

In certain non-limiting embodiments, the composition further comprisesother agents. These agents may include compositions designed to assistin reducing inflammation, aiding wound healing, promoting angiogenesisand/or vasculogenesis, and degrading collagen. Agents useful forreducing inflammation include, but are not limited to, anti-inflammatorycytokines such as interleukin-4 (IL-4), IL-6, IL-10, IL-11, and IL-13.Cytokines are cell-signaling molecules, which may be categorized aspeptides, proteins, or glycoproteins. IL-1 receptor antagonists (IL-1Ra)may also be included in the composition to reduce inflammation in thearea of treatment. In certain non-limiting embodiments, theanti-inflammatory agents may include free radical scavengers orantioxidants such as, without limitation, glutathione, selenium, vitaminE, vitamin C, and/or beta-carotene.

Agents useful for enhancing wound healing that may be included with thecomposition of the present invention include, but are not limited to,growth factors, collagenases, fullerenes and derivatives thereof,pseudopterosin-based compounds, histatin, anti-fibrotic compounds suchas tumor growth factor-beta inhibitors, analgesics, and the like. Inpreferred, non-limiting embodiments, the agent useful for wound healingis fibroblast growth factor (FGF).

Agents useful for enhancing angiogenesis and/or vasculogenesis that maybe included with the composition of the present invention include, butare not limited to, growth factors, matrix mealloproteinases (MMPs), andangiopoietins (Ang1 and Ang2). The growth factors useful in the presentcomposition include the vascular endothelial growth factor (VEGF) familyincluding VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental growth factor(PGF), and the FGF family (FGF-1 and FGF-2). In certain preferrednon-limiting embodiments, the agent useful for enhancing angiogenesisand/or vasculogenesis is selected from the VEGF family.

As Peyronie's disease is known to result from disorganized collagen, incertain preferred embodiments it is also useful to include a collagenasein the composition, to aid in the breaking down of disorganized collagenin the plaques associated with the disease. Collagenases are enzymesthat break the peptide bonds in collagen and aid in breaking down theprotein. Collagenases useful in the composition of the present inventionmay include MMPs and collagenase clostridial histolyticum (availablefrom Auxilium Pharmaceuticals under the trade name Xiaflex®).

The composition of the present invention may further include a biologicmatrix or growth scaffold on which the adipose tissue and stem cells arepresent. In some embodiments the matrix is a degradable matrix, suchthat after implantation, the matrix is slowly dissolved by the body'snatural processes. Such a scaffold may include collagen fibers orbundles, for example the Three Dimensional Collagen Composite Scaffoldavailable from Becton, Dickinson and Company. Other scaffolds may beformed from isolated components of ECM, or whole ECM. Such scaffolds areavailable, for example, from Kensey Nash under the trade name MesoBioMatrix™ and include porcine mesothelial ECM.

In addition, scaffolds made from synthetic materials may also be used inthe composition of the present invention. These scaffolds may beprovided by any known process familiar to those in the polymer arts. Thesynthetic scaffold may include a fabric that can be attached to the siteof interest by sutures or the like, or a may be held in place by anadhesive that is acceptable for use in humans. Such scaffolds can beformed into any suitable shape.

In a preferred, non-limiting embodiment, the composition is provided onan ECM scaffold. The ECM may be obtained from any commercial source. TheECM scaffold may be biodegradable or not, but preferably isbiodegradable. In such a non-limiting embodiment, the adipose tissue andat least one isolated stem cell may be disposed on the surface of thescaffold, or may be embedded within a three-dimensional scaffold.Formation of such scaffolds, through dissociation of tissue, digestion,lyophilization, and reconstitution, may be accomplished by any procedureknown to those of skill in the art.

In some non-limiting embodiments, the scaffold is a fibrin scaffold thatis injectable, for example through the method also provided herein.Injectable fibrin scaffolds are known in the art, for example, a grouphas seen growth of cardiac cells following implantation of ADRCs on afibrin scaffold via injection. Zhang et al., Preservation of the cardiacfunction in infracted rat hearts by the transplantation ofadipose-derived stem cells with injectable fibrin scaffolds. Exper.Biol. & Med. 2010; 235: 1505-1515.

Another aspect of the current invention provides for a method oftreating an individual having a penile defect. The method comprisesproviding a composition of at least one isolated stem cell, andimplanting the composition within the penis of the individual. Themethod may be used for treatment of erectile dysfunction (ED) andPeyronie's disease, among other defects or disorders.

In a non-limiting embodiment, the implanting step comprises providing abiological scaffold or matrix comprising the composition. Examples ofsuitable biological scaffolds are known to those skilled in the art. Insome embodiments the matrix is a degradable matrix, such that afterimplantation, the matrix is slowly dissolved by the body's naturalprocesses.

In a further preferred embodiment, the composition is provided on an ECMscaffold, for example, from Kensey Nash under the trade name MesoBioMatrix™, and that scaffolding is then implanted into the subject. TheECM scaffold may be biodegradable or not.

In yet another non-limiting embodiment, the composition is implantedinto a subject by injection. The pellet of cells obtained by the methodprovided herein is resuspended in a suitable solution and injected intoappropriate areas of the body.

In preferred, non-limiting embodiments, the implantation is directedtoward the subject's penis. Implantation into the penis may be at anysuitable location or anatomy for addressing the particular defect.Implantation may be of the composition, including in some embodimentsadipose tissue and/or a scaffold or matrix, by injection of a solution,or by any other method known for introducing stem cells to a target. Innon-limiting embodiments, injection occurs at the target site and is inat least 50 microliter volumes delivered in a needle of suitable gaugefor the anatomy.

In further preferred, non-limiting embodiments, the implantation isdirected towards at least one of the subject's corpora cavernosa, Inpreferred, non-limiting embodiments, a solution comprising thecomposition of isolated stem cells is injected directly into or aroundat least one of the corpora cavernosa. In preferred, non-limitingembodiments, a solution comprising the composition of isolated stemcells is injected directly into scar tissue present on the penis of anindividual with Peyronie's disease. In additional non-limitingembodiments, the composition is injected or infused into any artery thatfeeds penile tissue. In certain non-limiting embodiments, thecomposition is injected or infused into the corporal arteries orinternal pudendal arteries.

In another non-limiting embodiment, the composition is implanted into oraround the subject's tunica albuginea. Stem cells, such as ADRCs in anon-limiting embodiment, stimulate the growth of human tissues. Woundedtissue is most often replaced by scar tissue, such as that present inPeyronie's disease. This scar tissue is characterized in the skin bydisorganized collagen structure and irregular vascular structure. Stemcells reorganize collagen and induce vasculogenesis, aiding in woundhealing, such as in wounds present in Peyronie's disease. See Thomas J.Stem cells repair damaged corneas in mice. Healing Well. Apr. 10, 2009,available at http://news.healingwell.com/index.php?p=news1&id=625948.

Proliferation and/or differentiation of cells may be accomplished beforeor after transplantation, and in various combinations of in vitro or invivo conditions, including (1) proliferation and differentiation invitro, then transplantation, (2) proliferation in vitro,transplantation, then further proliferation and differentiation in vivo,and (3) proliferation in vitro, transplantation and differentiation invivo. Those skilled in the art can follow standard methodology totransform the stem cells of the current invention into a desired celltype or engineered construct for use in implantation for the purposesdescribed herein.

EXAMPLES Example 1—Treatment of Peyronie's Disease

The composition of the present invention is administered to a populationhaving Peyronie's disease. Peyronie's disease is an ailment involvingthe growth of fibrous scar tissue, or plaques, in the soft tissue of thepenis, which results in abnormal bending of the organ. The etiology ofthe plaques is not known. These plaques grow in the tunica albuginea, aregion of connective tissue that is part of the more general connectivetissue of Buck's fascia. The tunica albuginea is an area surrounding thecorpora cavernosa. The disease results in pain, ED, and alteration ofpenis shape, and may be caused by disorganized collagen fibers.Treatments for the disease currently include administration ofcorticosteroids to the plaques, radiation therapy, and vitamin EPharmaceuticals for treatment exist; however, the use of medicaments isconsidered controversial.

Prior to administration of the composition of the present invention,baseline measurements are taken to assess the state of health of thesubjects. These assessments include the following:

Subjects will have peak penile artery velocity in the corporal arteriesmeasured with penile doppler and ultrasound. Penile doppler is astandard tool for the evaluation of ED, and is less invasive thantypical means for measuring artery velocity, angiography with selectiveinternal iliac angiography. Penile Doppler may be performed with a highfrequency transducer (7.5-9.0 MHz), and at least the inner diameter ofthe cavernosal artery, baseline peak systolic velocity, and enddiastolic velocity are measured. A normal value for inner diameter ofthe cavernosal artery is 0.3-0.5 mm. In a flaccid state, monophasic flowshould be seen in an individual without Peyronie's disease. In an erectstate, a velocity of greater than 30 cm/sec should be observed in anindividual without ED, which is often a symptom of Peyronie's disease.

In addition to penile artery velocity, the size of fibrous plaquespresent on the penis are also assessed prior to the initiation of anytreatment. This may be done with ultrasound, at which time the grade ofPeyronie's may be assigned to the subject. Peyronie's grades are givenaccording to the following: Grade 1 (plaque less than 0.3 cm), Grade 2(greater than 0.3 cm and less than 1.5 cm), and Grade 3 (greater than1.5 cm, or two plaques greater than 1 cm).

Angle of penile curvature, a hallmark of Peyronie's disease, is alsomeasured prior to initiation of any treatment. Curvature may be assigneda grade according to the following, based on the Kelami classificationsystem: Grade 1 (curvature of 30 degrees or less), Grade 2 (curvature of30 degrees to 60 degrees), and Grade 3 (curvature of greater than 60degrees).

A penile rigidity test is also performed prior to any initiation oftreatment. This test may be done as a nocturnal penile tumescence (NPTtest) or an intracavernosal injection test. In an NPT test, thefrequency and quality of an erection during sleep is assessed by eitherplacing a ring-like device around the subject's penis, or by use of anelectronic monitoring device. The ring-like device is a simplemechanical device consisting of plastic films, which shear at certainpressures, for example when an erection provides sufficient pressure onthe ring to break the film. The electronic measurement includes a devicethat measures frequency, temporal length, and rigidity of erectionsduring sleep. This is a preferred means for measuring rigidity. Thesevariations of the NPT test may be conducted in a polysomnography orother sleep lab, or at the subject's home. The NPT test is usuallyperformed over two consecutive nights for accuracy.

Another form of the rigidity test, the intracavernosal injection test,involves injection of alprostadil (a formulation of prostaglandin E1available under the trade names Edex® from actient pharmaceuticals andCaverject® from Pfizer) or Tri-Mix (a mixture of Prostaglandin E1,Phentolamine, and Papaverine) into the base of the penis, which causesan erection through its vasodilator properties. Following the injection,fullness and length of the erection are measured. The test may berepeated as necessary for increased accuracy. The length andcircumference of each subject's penis is also assessed prior totreatment.

Subjects are also given the International Index of Erectile Function(IIEF) Questionnaire prior to any initiation of treatment. Thisquestionnaire asks subjects to rate various parameters relating to ED byanswering fifteen questions, and assigns point values from zero to fiveto each answer. A score of 25-30 indicates no ED, and a score of 0-6indicates severe ED. A continuum of moderate to mild ED exists between ascore of 7 and 24. A shortened version of the IIEF, the IIEF-5 may beadministered. In this short form, five questions are to be answered, anda score of 22-25 means that the subject does not have ED, and a score of5-7 means the subject has severe ED. A continuum of moderate to mild EDexists between a score of 7 and 22.

Following baseline measurements of the above variables, the compositionincluding adipose tissue and stem cells is administered to the subjectby implantation in the penis. This implantation is by surgical incisionand implantation with a biodegradable scaffold having adipose tissue andstem cells present therein, by injection of the composition on aninjectable fibrin scaffold, or injection of adipose tissue and stem cellalone, or with an injectable fibrin scaffold. Various groups includingother elements such as growth factors, anti-inflammatories,antioxidants, and collagenases are included. Injections are made intothe tunica albuginea surrounding the corpora cavernosa and/or thecorpora cavernosa themselves, any artery feeding penile tissue, or thecorporal arteries or internal pudendal arteries. Repeated injections maybe necessary.

Following administration of the composition, follow-up observations,including each of the aforementioned variables (arterial velocity,penile plaque size, penile curvature, IIEF score, and rigidity testing)are collected at three months, six months, and twelve months. Increasedvelocity, decreased plaque size, decreased curvature, increased IIEFscore, and increased rigidity are expected. Shortening of time forimprovement is expected in those receiving collagenase as well.

Example 2—Treatment of Erectile Dysfunction

The composition of the present invention is administered to a populationhaving ED. ED is an ailment in which a male is unable to achieve orsustain an erection suitable for sexual intercourse. A number of factorsare believed to play a role in or be directly responsible for ED,including obesity, blood pressure, chronic illnesses such as diabetes,poor blood flow to the penis, smoking tobacco, alcoholism, andside-effects of other medications.

Treatments for ED currently include cessation of potential causes suchas smoking tobacco and consumption of alcohol, hormone (testosterone)replacement, surgery, and administration of pharmaceuticals such asvardenafil, tadalafil, and sildenafil. Some of these pharmaceuticals arecontroversial for their incompatibility with nitrate drugs, and fortheir unwanted side-effects, such as effects on vision (blurring, lossof vision) and priapism.

Prior to administration of the composition of the present invention,baseline measurements are taken to assess the state of health of thesubjects. These assessments include the following:

Subjects will have peak penile artery velocity in the corporal arteriesmeasured with penile doppler and ultrasound. Penile doppler is astandard tool for the evaluation of ED, and is less invasive thantypical means for measuring artery velocity, angiography with selectiveinternal iliac angiography. Penile Doppler may be performed with a highfrequency transducer (7.5-9.0 MHz), and at least the inner diameter ofthe cavernosal artery, baseline peak systolic velocity, and enddiastolic velocity are measured. A normal value for inner diameter ofthe cavernosal artery is 0.3-0.5 mm. In a flaccid state, monophasic flowshould be seen. In an erect state, a velocity of greater than 30 cm/secshould be observed in an individual without ED.

A penile rigidity test is also performed prior to any initiation oftreatment. This test may be done as a nocturnal penile tumescence (NPTtest) or an intracavernosal injection test. In an NPT test, thefrequency and quality of an erection during sleep is assessed by eitherplacing a ring-like device around the subject's penis, or by use of anelectronic monitoring device. The ring-like device is a simplemechanical device consisting of plastic films, which shear at certainpressures, for example when an erection provides sufficient pressure onthe ring to break the film. The electronic measurement includes a devicethat measures frequency, temporal length, and rigidity of erectionsduring sleep. This is a preferred means for measuring rigidity. Thesevariations of the NPT test may be conducted in a polysomnography orother sleep lab, or at the subject's home. The NPT test is usuallyperformed over two consecutive nights for accuracy.

Another form of the rigidity test, the intracavernosal injection test,involves injection of alprostadil (a formulation of prostaglandin E1available under the trade names Edex® from actient pharmaceuticals andCaverject® from Pfizer) or Tri-Mix (a mixture of Prostaglandin E1,Phentolamine, and Papaverine) into the base of the penis, which causesan erection through its vasodilator properties. Following the injection,fullness and length of the erection are measured. The test may berepeated as necessary for increased accuracy. The length andcircumference of each subject's penis is also assessed prior totreatment.

Subjects are also given the International Index of Erectile Function(IIEF) Questionnaire prior to any initiation of treatment. Thisquestionnaire asks subjects to rate various parameters relating to ED byanswering fifteen questions, and assigns point values from zero to fiveto each answer. A score of 25-30 indicates no ED, and a score of 0-6indicates severe ED. A continuum of moderate to mild ED exists between ascore of 7 and 24. A shortened version of the IIEF, the IIEF-5 may beadministered. In this short form, five questions are to be answered, anda score of 22-25 means that the subject does not have ED, and a score of5-7 means the subject has severe ED. A continuum of moderate to mild EDexists between a score of 7 and 22.

Following baseline measurements of the above variables, the compositionincluding adipose tissue and stem cells is administered to the subjectby implantation in the penis. This implantation is by surgical incisionand implantation with a biodegradable scaffold having adipose tissue andstem cells present therein, by injection of the composition on aninjectable fibrin scaffold, or injection of adipose tissue and stem cellalone. Various groups including other elements such as growth factors,anti-inflammatories, antioxidants, and collagenases are included.Injections are made into the tunica albuginea surrounding the corporacavernosa and/or the corpora cavernosa themselves, any artery feedingpenile tissue, or the corporal arteries or internal pudendal arteries.Repeated injections may be necessary.

Following administration of the composition, follow-up observations,including each of the aforementioned variables (arterial velocity,rigidity testing, and IIEF score) are collected at three months, sixmonths, and twelve months. Increased velocity, increased IIEF score, andincreased rigidity are expected.

While the present invention has been described in connection with thepreferred embodiments, it is to be understood that other similarembodiments may be used or modifications and additions may be made tothe described embodiments for performing the same function of thepresent invention without deviating therefrom. Therefore, the presentinvention should not be limited to any single embodiment, but ratherconstrued in breadth and scope in accordance with the recitation of theappended claims.

Variations in the present invention are possible in light of thedescription of it provided herein. While certain representativeembodiments and details have been shown for the purpose of illustratingthe subject invention, it will be apparent to those skilled in this artthat various changes and modifications can be made therein withoutdeparting from the scope of the subject invention. It is, therefore, tobe understood that changes can be made in the particular embodimentsdescribed which will be within the full intended scope of the inventionas defined by the following appended claims.

What is claimed is:
 1. A method of treating a subject with a peniledefect comprising the steps of: providing a composition comprising atleast one isolated stem cell; and implanting the composition within thepenis of a subject in need of treatment for a penile defect, wherein theat least one stem cell is a mesenchymal stem cell, wherein themesenchymal stem cell is an ADRC
 2. The method according to claim 1,wherein the adipose tissue and ADRC are derived from the subject.
 3. Themethod of claim 1, wherein the composition further comprises abiological scaffolding, wherein the scaffolding is a fibrin scaffolding.4. The method of claim 1, wherein the composition is injected into oraround the tunica albuginea.
 5. The method of claim 1, wherein thedefect is Peyronie's disease.
 6. The method of claim 1, wherein thecomposition comprises at least one additional agent.
 7. The method ofclaim 6, wherein the agent promotes at least one of wound healing,angiogenesis, vasculogenesis, and degradation of collagen.
 8. The methodof claim 6, wherein the agent is an anti-inflammatory or an antioxidant.9. The method of claim 1, wherein the at least one stem cell is aplacental stem cell.
 10. The method of claim 3, wherein the scaffoldcomprises collagen.